Antioxidant defense against antidepressants in C6 and 1321N1 cells

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Abstract

The effects of pretreatment with the antioxidants reduced glutathione (GSH), ascorbate (ASC), Trolox (TROL), and combined ascorbate and Trolox (ASC/TROL) exposure on the acute (24 h) toxicities (EC50 value) of the antidepressants amitriptyline, imipramine (tricyclic antidepressants), fluoxetine (a selective serotonin reuptake inhibitor; SSRI), and tranylcypromine (a monoamine oxidase inhibitor; MAOI) were determined in the rat (C6) glioma and human (1321N1) astrocytoma cell lines using the neutral red uptake assay. The effects of pretreatment with buthionine-[S,R]-sulfoximine (BSO), and manipulation of intracellular cyclic AMP (cAMP) using isoproterenol (β-receptor agonist), 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor), and dibutyryl cyclic AMP (dBcAMP; cAMP analogue) on antidepressant toxicity were also determined. Protective responses were observed after antioxidant treatments and manipulation of cAMP in both C6 cells pretreated with dBcAMP (+dBcAMP) and 1321N1 cells not pretreated with dBcAMP (−dBcAMP), with a few exceptions in 1321N1 cells (−dBcAMP). Some protective responses occurred in C6 cells (−dBcAMP) and 1321N1 cells (+dBcAMP) after isoproterenol and combined IBMX/isoproterenol pretreatment but not after just IBMX pretreatment. Pretreatment with BSO enhanced toxicity with the exception of fluoxetine. The antidepressants caused increases in intracellular GSH in the C6 cells at subcytotoxic concentrations, with decreases in GSH occurring at higher concentrations. Cytotoxicity of the antidepressants may be partly mediated through oxidative stress with alterations in signal transduction pathways.

Introduction

The mechanisms underlying the actions of antidepressants are not clearly understood. The latency of clinical response may not be attributable to their acute pharmacological actions and considerable evidence suggests that the signalling pathways which regulate gene transcription through the cAMP and Ca2+ response element may be affected at multiple levels (for review see [1]). The downregulation of 5HT2 and β-adrenergic receptors and blocking of voltage-dependent calcium channels, together with alterations in G-protein activities are well documented changes [2], [3]. However, most antidepressants also possess significant toxicities and the incidence of overdosing is high [4]. Despite the widely documented neurological impairments, which may be caused by antidepressants, there are no available data on the underlying processes.

A considerable amount of information on antidepressants has been obtained from in vitro studies. These include pharmacological, biochemical and toxicological studies on both neurons and astrocytes. The astrocytes in particular have attracted attention because both short and relatively long-term culture studies can be made which may more closely reflect the effects of acute or chronic treatments. For example, some of the earlier studies demonstrated that chronic exposure of primary mouse astrocytes to antidepressants downregulated adenylyl cyclase linked β-adrenergic receptors with a time course which paralleled that seen in vivo in animal brain homogenates and the therapeutic improvement seen in depressed patients [5]. The great potential importance of astrocytes in the therapeutic effects of antidepressants and other neuroactive drugs was thus established [6].

There is now substantial evidence that the astrocytes have key protective capacities against a range of potentially damaging effects in the CNS and that these capacities may be increased in reactive states [7], [8]. Among these, the antioxidant defence system appears especially effective and becomes accentuated following reactive gliosis [8], [9].

In a recent study, we analysed the effects of several antidepressants in cultured rat C6 glioma and human (132#1N1) astrocytoma cell lines using a number of astrocyte-specific, metabolic and cytotoxic methods [10]. This work demonstrated that there was an overall good correlation between EC50 values in the cell lines and LD50 values (RTECS listed) for the antidepressants tested, with several substances categorised as extremely toxic. However, it also showed that the antidepressants could initiate activation-type responses at subcytotoxic doses. These were evident as increases in GFAP levels with increased branching of cell processes and increases in MTT conversion and neutral red uptake. Other workers have previously shown that treatment of astrocyte cultures with β-adrenergic agonists or dibutyryl cyclic AMP (dBcAMP) initiate biochemical and morphological changes which have been used to model aspects of reactive astrocytes in vitro [7], [11], [12], [13], [14]. We have also found that these changes are associated with increased protective capacities against a number of toxic substances [15].

The purpose of the present work was to assess the protective capacities of astrocytes against the acute effects of antidepressants using two astrocytes cell lines. Cells were exposed to antidepressants and antioxidant levels manipulated by drug treatments. In some experiments, the levels of cyclic AMP (cAMP) were altered to induce activation-type responses. The work shows that the cells’ defence systems protected against damage caused by the antidepressants and that the damage might be caused by oxidative stress. Modifications of the cAMP levels indicated that activation-type responses might confer increased protective capacity.

Section snippets

Materials

All materials were obtained from Sigma unless otherwise stated. C6 and 1321N1 cells were obtained from the European collection of animal cell cultures at Porton down, Salisbury, UK.

Cell culture methods

C6 and 1321N1 cells were subcultured from stock flasks according to our previous descriptions [10]. In brief, cells were washed in Hanks balanced salt solution (HBSS, 3×5 ml), trypsinised (1 ml trypsin-EDTA), and diluted with 9 ml of astrocyte growth medium (AGM). The AGM used was DMEM supplemented with 10% (v/v)

Effects of antioxidants

The EC50 value changes in C6 and 1321N1 cells after antioxidant treatment are summarised in Table 1, Table 2. There were significant protective shifts after GSH treatment in C6 cells (pretreated with dBcAMP), this was greatest for fluoxetine (Table 1). Buthionine-[S,R]-sulfoximine (BSO) which depletes intracellular glutathione levels enhanced the toxicity of some of the antidepressants (negative shifts in EC50 value) in C6 cells (+dBcAMP), although these changes were not significant. In

Discussion

The antidepressants were chosen for study because they represent the major classes of antidepressants (i.e. the monoamine oxidase inhibitors, tricyclics and selective serotonin re-uptake inhibitors) and are also the widely prescribed antidepressants of the classes. In contrast to the extensive literature on the pharmacological and biochemical processes which may underlie the beneficial therapeutic effects of the antidepressants, very little is known about the ways they may initiate changes in

Acknowledgements

This work was funded by the Science Research Institute (Salford University). The authors would like to thank Dr G. Hide for statistical assistance.

References (32)

  • D.A.G. Mickle

    Myocardial salvage with Trolox and ascorbic acid for an acute evolving infarction

    Ann. Thorac. Surg.

    (1989)
  • H.K. Kimelberg

    Receptors on astrocytes — what possible functions?

    Neurochem. Int.

    (1995)
  • S. Caccia et al.

    Pharmacokinetic and pharmacodynamic significance of antidepressant drug metabolites

    Pharmacol. Res.

    (1992)
  • P.E. Stokes et al.

    Fluoxetine tenth anniversary update: the progress continues

    Clin. Therapeutics

    (1997)
  • M. Schwaninger et al.

    Progress in defining the mechanism of action of antidepressants. Across receptors and into gene transcription

    CNS Drugs

    (1997)
  • G.B. Baker et al.

    Effects of long-term administration of antidepressants and neuroleptics on receptors in the central nervous system

    Cell. Mol. Neurobiol.

    (1989)
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    Present address: Development DMPK II Alderley, AstraZeneca Pharmaceuticals, Alderley Park, Alderley Edge, Macclesfield SK1O 4TG, UK.

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